The goal of the proposed research is to uncover the roles(s) in viral replication of a host cell protein recently identified as being VPg unlinkase for the picornavirus family. Picornaviruses are small, positive-strand RNA viruses that cause a wide range of diseases in humans and animals, with significant health and economic repercussions. The importance of these viruses extends beyond their public health implications; as the first mammalian viruses discovered, the study of picornaviruses has had a major impact on viral molecular biology and led to important findings related to cellular receptors for viruses, infectious cDNAs for RNA viruses, internal ribosome entry site (IRES)-driven translation, anti-viral responses of mammalian cells, the maturation of proteins synthesized as polyprotein precursors, chemical synthesis of a virus, and a host of other important research areas. A major theme that has emerged during the last decade is how these viruses with a very limited coding capacity utilize and/or modify host cell functions to complete their replication cycles. One such function is the cellular activity, VPg unlinkase, that removes the small viral peptide (VPg) from the 5' end of picornavirus genomic RNAs by cleaving a protein-nucleotidyl bond prior to or after the onset of viral protein synthesis. The PI's laboratory has recently identified VPg unlinkase from HeLa cells as tyrosyl-DNA phosphodiesterase 2 (TDP2), a host enzyme involved in DNA repair, cell signaling, and transcriptional regulation. This proposal aims to characterize the activity of TDP2/VPg unlinkase in cells infected by enteroviruses (poliovirus or coxsackievirus) and by the closely related human rhinovirus to determine how viral replication is impacted when this activity is down-regulated during infection. The specific aims are: (i) Down-regulate TDP2/VPg unlinkase activity to define its function(s) in picornavirus replication, and (ii) Define the cellular relocalization pathway and protein-binding partners of TDP2/VPg unlinkase in picornavirus-infected cells. The proposed experimental plan will examine the hypothesis that picornaviruses employ TDP2/VPg unlinkase activity to regulate the fate of viral RNAs to carry out specific functions in translation, RNA replication, or assembly into infectious virus particles. Results from the proposed studies may reveal TDP2/VPg unlinkase activity as a novel target for anti-viral therapy directed at human rhinovirus, coxsackievirus, hepatitis A virus, and other picornaviruses responsible for significant human disease sequelae.